ROLE OF PROTEIN-KINASE-C-ALPHA IN ENDOTHELIN-1 STIMULATION OF CYTOSOLIC PHOSPHOLIPASE A(2) AND ARACHIDONIC-ACID RELEASE IN CULTURED CAT IRIS SPHINCTER SMOOTH-MUSCLE CELLS

We have investigated the role and mechanism of protein kinase C (PKC) isoforms in endothelin-1 (ET-1)-induced arachidonic acid (AA) release in cat iris sphincter smooth muscle (CISM) cells. ET-1 increased AA re lease in a concentration (EC50 = 8 nM) and time-dependent (t(1/2) = 1. 2 min) manner. Cytosolic phospholipase A(2) (cPLA(2)), but not phospho lipase C (PLC), is involved in the liberation of AA in the stimulated cells. This conclusion is supported by the findings that ET-1-induced AA release is inhibited by AACOCF(3), Quinacrine and manoalide, PLA(2) inhibitors, but nor by U-73122, a PLC inhibitor, or by RHC-80267, a d iacylglycerol lipase inhibitor. A role for PKC in ET-l-induced AA rele ase is supported by the findings that the phorbol ester, PDBu, increas ed AA release by 96%, that prolonged treatment of the cells with PDBu resulted in the selective down regulation of PKC alpha and the complet e inhibition of ET-l-induced AA release, and that pretreatment of the cells with staurosporine or RO 31-8220, PKC inhibitors, blocked the ET -l-induced AA release. Go-6976, a compound that inhibits PKC alpha and beta specifically, blocked ET-l-induced AA release in a concentration -dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 mu M), a specific activator of PKC alpha, beta, and gamma induced a 150% incr ease in AA release. Treatment of the cells with ET-1 caused significan t translocation of PKC alpha, but not PKC beta, from cytosol to the pa rticulate fraction. These results suggest that PKC alpha plays a criti cal role in ET-1-induced AA release in these cells. Immunochemical ana lysis revealed the presence of cPLA(2), p42(mapk) and p44(mapk) in the CISM cells. The data presented are consistent with a role for PKC alp ha, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cP LA(2) activation and AA release in ET-l-stimulated CISM cells since: ( i) the PKC inhibitor, RO 31-8220, inhibited ET-l-induced AA release, c PLA(2) phosphorylation and cPLA(2) activity, but had no inhibitory eff ect on p42/p44 MAPK activation, (ii) genistein, a tyrosine kinase inhi bitor, inhibited ET-l-stimulated MAPK activity but had no inhibitory e ffect on AA release in the ET-l-stimulated cells. We conclude that in CISM cells, ET-1 activates PKC alpha, which activates cPLA(2), which l iberates AA for prostaglandin synthesis. (C) 1998 Elsevier Science B.V . All rights reserved.